Mechanism of action of novel subunit-selective NMDA receptor modulators

描述（由申请人提供）：n -甲基- d-天冬氨酸选择性谷氨酸受体（NMDA受体）是配体门控离子通道，介导脑和脊髓的兴奋性突触传递。NMDA受体是由2个甘氨酸结合NR1亚基和2个谷氨酸结合NR2亚基组成的异聚体，有4个家族成员（NR2A，B，C，D）。NMDA受体参与许多正常的脑功能，如神经元发育、学习、记忆，除了在中风、癫痫和神经精神疾病等神经病理条件中发挥作用外。自首次描述以来，nr2b选择性NMDA受体拮抗剂伊芬普罗地尔及其许多类似物已被用于大量研究，探索该亚基在脑功能的几乎每个方面的作用，包括行为、认知、突触可塑性、神经元发育和神经病理学。然而，对于NR2A-、NR2C-或nr2d受体，尚无高选择性拮抗剂被描述。因此，我们开发了一种检测方法来鉴定含有NR2C-和nr2d -的NMDA受体的非竞争性变构调节剂，并随后筛选了约60,000种化合物作为新的亚单位选择性拮抗剂和增强剂。结果是鉴定出两类结构独特的化合物，与含有NR2A或nr2b的受体或AMPA/kainate受体相比，它们是NR1/NR2C和NR1/NR2D的100-500倍选择性抑制剂。我们还发现了两种结构上不同的增强剂，它们对NR2C/ d -含受体具有选择性。这些新的非竞争性变构调节剂为研究NR2C/D亚基在正常脑功能和神经系统疾病中的作用提供了一个突破性的机会。本建议使用这些亚单元选择性调制器解决3个问题。1. NR2C/ d选择性抑制剂和增强剂的结构决定因素是什么？我们将利用位点定向诱变，利用NR2A/B和NR2C/D之间的序列差异，确定介导三类NR2C/D选择性增强剂和抑制剂作用的关键残基。2. NR2C/D选择性抑制剂和增强剂的作用机制是什么？我们将分析在电压钳下记录的宏观和单通道电流，以确定非竞争性NR2C/NR2D抑制剂以及NR2C/NR2D增强剂的作用机制。3. NR2C/D调节剂如何改变突触信号和神经元兴奋性？我们将在脑切片中评估皮质-丘脑下神经元突触中含有NR2C/ d的NMDA受体和海马间神经元传入兴奋突触中含有NR2C/ d的NMDA受体的抑制和增强作用。实验将测试NR2C/D调节剂存在时突触对刺激序列的反应。我们还将使用这些新的药理学工具来研究神经元间和丘脑下神经元的兴奋性和峰值频率是否可以通过抑制或增强NR2C/ d -含受体而改变。

DESCRIPTION (provided by applicant): N-methyl-D-aspartate-selective glutamate receptors (NMDA receptors) are ligand-gated ion channels that mediate excitatory synaptic transmission in the brain and spinal cord. NMDA receptors are heteromultimers comprised of two glycine-binding NR1 subunits and two glutamate-binding NR2 subunits, of which there are four family members (NR2A,B,C,D). NMDA receptors are involved in many normal brain functions such as neuronal development, learning, memory, in addition to their roles in neuropathological conditions such as stroke, epilepsy, and neuropsychiatric disorders. Since its first description, the NR2B-selective NMDA receptor antagonist ifenprodil and its many analogues have been used in a multitude of studies exploring the role of this subunit in virtually every aspect of brain function including behavior, cognition, synaptic plasticity, neuronal development, and neuropathology. However, no highly selective antagonists have yet been described for NR2A-, NR2C-, or NR2D-containing receptors. We therefore developed an assay to identify non-competitive allosteric modulators of NR2C- and NR2D-containing NMDA receptors, and subsequently screened ~60,000 compounds for new subunit-selective antagonists and potentiators. The result was the identification of two structurally unique classes of compounds that are 100-500 fold selective inhibitors of NR1/NR2C and NR1/NR2D compared to NR2A- or NR2B-containing receptors or AMPA/kainate receptors. We also identified two structurally distinct classes of potentiators that are selective for NR2C/D-containing receptors. These new non-competitive allosteric modulators represent a breakthrough opportunity to study the role of the NR2C/D subunits in normal brain function and in neurological diseases. This proposal addresses 3 questions using these subunit-selective modulators. 1. What are the structural determinants of NR2C/D-selective inhibitors and potentiators? We will utilize site-directed mutagenesis, exploiting sequence differences between NR2A/B and NR2C/D, to identify key residues that mediate the actions of three classes of NR2C/D-selective potentiators and inhibitors. 2. What is the mechanism of action of NR2C/D selective inhibitors and potentiators? We will analyze macroscopic and single channel currents recorded under voltage clamp to define the mechanism of action of the non-competitive NR2C/NR2D inhibitors as well as NR2C/NR2D potentiators. 3. How do NR2C/D modulators alter synaptic signaling and neuronal excitability? We will evaluate the effect of inhibition and potentiation of NR2D-containing NMDA receptors at the cortical-subthalamic neuron synapse and NR2C/D-containing NMDA receptors at afferent excitatory synapses onto hippocampal interneurons in brain slices. Experiments will test the synaptic response to stimulus trains in the presence of NR2C/D modulators. We will also use these new pharmacological tools to investigate whether interneuron and subthalamic neuron excitability and spiking frequency can be altered through inhibition or potentiation of NR2C/D-containing receptors.